Abstract
The present disclosure provides molecules which modulate cell growth. These molecules include those that bind sialic acid which may find application in the treatment and/or prevention of cell proliferation and/or differentiation disorders, cancer and/or it's migration and/or spread.
Claims
1. A method of treating cancer, said method comprising administering a sialic acid binding molecule to a subject in need thereof, wherein the sialic acid binding molecule is a multimeric carbohydrate binding module (CBM).
2. The method of claim 1, wherein the cancer is lung cancer, cervical cancer, colon cancer and/or breast cancer.
3. The method of claim 1, wherein the multimeric carbohydrate binding module comprises one or more family 40 carbohydrate binding module(s).
4. The method of claim 1, wherein the multimeric carbohydrate binding module comprises a sialic acid binding domain of Vibrio cholerae NanH sialidase and/or a sialic acid binding domain of Streptococcus pneumoniae NanA sialidase.
5. The method of claim 4, wherein the Vibrio cholerae NanH sialidase comprises the amino acid sequence of SEQ ID NO: 1 or 2.
6. The method of claim 4, wherein the Streptococcus pneumoniae NanA sialidase comprises the amino acid sequence of SEQ ID NO: 3 or 4.
7. The method of claim 1, wherein the multimeric CBM comprises two or more sialic acid binding domains of Vibrio cholerae NanH sialidases and/or two or more sialic acid binding domains of Streptococcus pneumoniae NanA sialidases.
8. The method of claim 1, wherein the multimeric CBM comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 sialic acid binding domains of Vibrio cholerae NanH sialidases and/or Streptococcus pneumoniae NanA sialidases.
9. The method of claim 1, wherein the multimeric CBM is a combination of four sialic acid binding domains of Vibrio cholerae NanH sialidase (Vc4CBM), a combination of two sialic acid binding domains of Vibrio cholerae NanH sialidase and a trimerisation domain (Vc2CBMTD), and/or a combination of two sialic acid binding domains of Streptococcus pneumoniae NanA sialidase and a trimerisation domain (Sp2CBMTD).
Description
DETAILED DESCRIPTION
(1) The present invention will now be described in detail by reference to the following Figures which show:
(2) FIG. 1. Building blocks of the multivalent CBM forms and their affinities for sialic acid. a, VcCBM, residues 25-216 of the V. cholerae sialidase (PDB:1w0p) with α-2,3-sialyllactose drawn as spheres. b, SpCBM, residues 121-305 of S. pneumoniae NanA sialidase with α-2,3-sialyllactose (PDB:4c1w). c, TD, the trimerisation domain, residues 333-438, of the P. aeruginosa pseudaminidase (PDB:2w38) in rainbow colors; the other two monomers in single colors. d, Multivalent forms: their molecular weights, valencies and binding affinities for α2,3-sialyllactose as determined by surface plasmon resonance (SPR) at 25° C. (K.sub.D values for VcCBM, Vc2CBM and Vc3CBM had been reported previously (Connaris et al, 2009)). Tandem repeat CBMs, and oligomeric CBMs fused to TD are linked by a 5-amino linker (details in Connaris et al, 2014).
(3) FIG. 2: CBM dose analysis on A549 human lung carcinoma cells. Left panel: 10-fold serially diluted single dose (i.e. 400 μg to 0.04 μg per well) of Vc2CBMTD (top panel), and of Sp2CBMTD (bottom panel). The right panel is identical to the left, except that cells are given CBMs twice over three days. CBMs were diluted in serum-free DMEM and incubated with cells for 3 h before removing and replaced with DMEM/5% FCS for cell maintenance. Mock wells represent cells that are given PBS only in DMEM/5% FCS.
(4) FIG. 3: Wound healing assay on A549 human lung carcinoma cells. Cells were scratched to create an artificial wound before treatment with PBS (A), 100 μg/ml Sp2CBMTD (B), or 100 μg/ml Vc2CBMTD (C), in serum-free DMEM. Cells were left for 24 h, followed by actin staining to visualise cell cytoskeleton.
(5) FIG. 4: (A) The MTT assay absorbance indicating the metabolic activity of A549 human lung carcinoma cells after treatment with either 50 μg/ml of monomeric CBM-like domain (TcTs), or 5 μg/ml of hexameric CBMs (Vc2CBMTD or Sp2CBMTD) resuspended in serum-free DMEM or in serum-free DMEM with 2 mM sialyllactose (+Sia) for 24 h at 37 deg C., 5% CO.sub.2. (B) Cell morphology of treated vs PBS only cells (control) after 24 h.
(6) FIG. 5: A. Scratch assay data from a human lung cancer cell line (A549) using mCBM40s. Hexameric Vc2CBMTD and Sp2CBMTD were tested at 100 μg in a 250 μL volume representing a concentration of 400 μg/ml. Phase contrast images represent the wound healing assay after 1 h and 72 h, after cells were treated with PBS, Vc2CBMTD, or Sp2CBMTD, in serum-free DMEM. Yellow bar at bottom of each image indicate diameter of 400 μm. B. Graphs representing the percentage of wound healing (ii), and rate of wound closure (ii) for A549 cells in the absence and presence of mCBM40s after 72 h. Dunnett's multiple comparison test was performed on the data. **** represents a statistical significance p<0.0001.
(7) FIG. 6: A. Scratch assay data from a human cervical cancer cell line (HeLa) using mCBM40s. Hexameric Vc2CBMTD and Sp2CBMTD were tested at 100 μg in a 250 μL volume representing a concentration of 400 μg/ml. Fluorescent images represent the wound healing assay after 1 h and 72 h, after cells were treated with PBS, Vc2CBMTD, or Sp2CBMTD, in serum-free DMEM. Yellow bar at bottom of each image indicate diameter of 400 μm. B. Graphs representing the percentage of wound healing (ii), and rate of wound closure (ii) for HeLa cells in the absence and presence of mCBM40s after 72 h. Dunnett's multiple comparison test was performed on the data. **** represents a statistical significance p<0.0001.
(8) FIG. 7: Scratch assay data for lung (A549), cervical (HeLa), breast (MDA.MB.231 and MCF-7) and colon (SW480 and SW620) cancer cell types in the presence and absence of Sp2CBMTD 100 μg (400 μg/mL). (a) Graph representing percentage of A549 cell wound closure in the presence and absence of Sp2CBMTD 100 μg after 72 hrs. (b) Graph representing percentage of HeLa cell wound closure in the presence and absence of Sp2CBMTD 100 μg after 72 hrs. (c). Graph representing percentage of MDA.MB.231 cell wound closure in the presence and absence of Sp2CBMTD 100 μg after 72 hrs. (d). Graph representing percentage of MCF-7 cell wound closure in the presence and absence of Sp2CBMTD 100 μg after 72 hrs. (e). Graph representing percentage of SW620 cell wound closure in the presence and absence of Sp2CBMTD 100 μg after 72 hrs. (f). Graph representing percentage of SW480 cell wound closure in the presence and absence of Sp2CBMTD 100 μg after 72 hrs. All data normalised to control. Unpaired t test performed on data, statistical significance (p) was reported between control and Sp2CBMTD (p=0.0001) highlighted by ****.
(9) FIG. 8: Cell proliferation of HeLa cells in the presence and absence of hexameric CBM40s. Graph depicts cell number against time (h). Vc2CBMTD and Sp2CBMTD were tested at 100 μg in a 250 μL volume representing a concentration of 400 μg/ml. PBS served as control.
(10) FIG. 9: Scratch assay data from a lung cancer cell line (A549) and a cervical cancer cell line (HeLa) using a tetrameric CBM40. Vc4CBM tested at 100 μg in a 250 μL volume representing a concentration of 400 μg/mL. A. Graphs representing percentage (i), and rate (ii), of A549 cell wound closure in the presence and absence of Vc4CBM after 72 hrs. B. Graph representing percentage (i) and rate (ii) of HeLa cell wound closure in the presence and absence of Vc4CBM also after 72 hrs. Unpaired t test was performed on all data with statistical significance (p) reported between control and Vc4CBM (p=0.0001) as highlighted by control and Vc4CBM highlighted by ****.
(11) FIG. 10: Effect of component parts of engineered multimeric CBM40s on cell proliferation of cancer cell lines using a wound healing assay after 72 h. Monomeric (VcCBM, SpCBM) and multimeric (Vc2CBMTD and Sp2CBMTD) CBM40s were tested at 100 μg in a 250 μL volume representing a concentration of 400 μg/mL. A Images representing wound healing assay in A459 (a) and HeLa (b) cells after 1 h and 72 h, after treatment with PBS, SpCBM, or VcCBM in serum-free DMEM. Yellow bar at bottom of each image indicate diameter of 400 μm. B Graphs representing percentage (i) (ii), and rate (iii) (iv) of wound closure in the presence and absence of CBM after 72 h in A549 and HeLa cell lines, respectively. Dunnett's multiple comparison test was performed on the data and a statistical significance (p) of 0.0001 is highlighted by **** and a p of 0.05 is highlighted by *.
(12) FIG. 11: Effect of component parts of engineered multimeric CBM40s on cell proliferation of A459 (A) and HeLa (B) cell lines using a wound healing assay after 72 h. The trimerization domain (TD) was tested at 100 μg in a 250 μL volume representing a concentration of 400 μg/mL. (a) Graph representing percentage of A549 cell wound closure in the presence and absence of TD after 72 hrs. Unpaired t test performed on data, no statistical significance (p) was reported between control and TD (p=0.1201). (b) Graph representing percentage of HeLa cell wound closure in the presence and absence of TD after 72 hrs. Unpaired t test performed on data, no statistical significance (p) was reported between control and TD (p=0.1201). (c). Graph representing the rate of A549 cell wound closure in the presence and absence of CBM after 72 hrs. Unpaired t test performed on data, a p of 0.05 was reported between control and TD (p=0.0212) highlighted by *. (d). Graph representing the rate of HeLa cell wound closure in the presence and absence of TD after 72 hrs. Unpaired t test performed on data, a p of 0.05 was reported between control and TD (p=0.0338) highlighted by *.
(13) FIG. 12: Scratch assay data plus wash off for A549 cell line in the presence and absence of CBM after 72 hrs followed by a further 72 hrs after wash off. A one-way ANOVA and a Dunnett's multiple comparison test were performed on the data and a statistical significance (p) of 0.0001 is highlighted by ****. A p of 0.0002 is highlighted by ***. (a). The p of 0.0001, 0.0002 and 0.05 between the 72 hr control is represented by ****, *** and * respectively. (a). Graph representing percentage of A549 cell wound closure in the presence and absence of CBM after 72 hrs and 144 hrs after a CBM wash-off at the 72 hr time point (72 hrs after wash). (b). Graph representing the rate of A549 cell wound closure in the presence and absence of CBM after 72 hrs and 144 hrs after a CBM wash-off at the 72 hr time point (72 hrs after wash).
(14) FIG. 13: Chemotaxis assay data from a lung cancer cell line (A549). A549 cells seeded at 8.0×10.sup.5 cells/well. Fluorescence measured after 24 hrs background corrected. CBM tested at 100 μg in a 100 μL volume representing a concentration of 1 mg/ml. Dunnett's multiple comparison test was performed on the data and a statistical significance (p) of 0.0001 is highlighted by ****.
(15) FIG. 14: Agglutination assay adapted from Hwang et al (1974). Graphs showing the absorbance at 546 nm of A549 cells in the absence (control) and presence of mCBM40 (Vc4CBM, Vc2CBMTD and Sp2CBMTD all at 1 mg/mL) for 5 mins, 30 mins and 1 hr (A), and at 1 hr and 24 hrs (B). A PBS only control was also included. Graphs showing the absorbance (C), and the rate of absorbance (D) of A549 cells at 546 nm for every ten seconds over 20 mins in the absence (control) and presence of mCBM40s after incubation on ice for 24 hrs.
(16) FIG. 15: Caspase dependent apoptosis. Graph showing the normalised (background corrected) luminescence signal produced by luciferase following caspase cleavage of Caspase-Glo® 3/7 reagent in A549 cells in the presence and absence of CBMs for 4 hrs, 24 hrs and 72 hrs. A one-way ANOVA and a Dunnett's multiple comparison test were performed on the data and a statistical significance (p) of ≤0.05 is highlighted by *.
(17) FIG. 16: Cell viability. Graph showing the normalised (background corrected) fluorescence signal produced by cleavage of GF-AFC substrate by A549 live cell protease in the presence and absence of CBMs for 4 hrs, 24 hrs and 72 hrs. A one-way ANOVA and a Dunnett's multiple comparison test were performed on the data and a statistical significance (p) of ≤0.05 is highlighted by *.
(18) FIG. 17: Cell cytotoxicity data for lung cancer cell line A549 in the presence and absence of CBM. Graph showing the normalised (background corrected) fluorescence signal produced by cleavage of bis-AAF-R110 substrate by A549 dead cell protease in the presence and absence of CBMs for 4 hrs, 24 hrs and 72 hrs. A one-way ANOVA and a Dunnett's multiple comparison test were performed on the data and a statistical significance (p) of ≤0.05 is highlighted by *.
EXAMPLE 1
(19) In an in vitro mCBM40 dosing experiment using confluent A549 human lung carcinoma cells and high concentrations of Sp2CBMTD (400-800 μg), little or no cell growth or activity was observed after 3 days. This was noted as the colour of the culture medium (DMEM/5% FCS), which would change colour as it is utilised, remained the same (see FIG. 2).
(20) This observation led to the hypothesis that our engineered sialic acid-recognising CBMs might affect cell (including cancer cell) proliferation and migration by targeting sialylated host cell receptors.
(21) To further support this hypothesis a wound scratch assay to determine cell migration and proliferation and a cell viability (MTT) assay were performed on A549 human lung carcinoma cells. These cells were treated with CBMs for 24 h. The results were compared to control cells that were given PBS only.
(22) As seen in FIG. 3, little or no wound healing was observed in CBM-treated wells compared to PBS after 24 h. PBS-treated cells demonstrated a scratch diameter of 250 μm with lone cells migrating into the artificial wound after 24 h. In contrast, scratch diameters for both CBM-treated cells were roughly 710 μm, with changes in cell morphology likely associated with cell death or cell cycle arrest for the Vc2CBMTD-treated cells. There is also poor cell migration into the wound by Sp2CBMTD-treated A549 cells. Interestingly, the formation of filopodia was observed with Sp2CBMTD, a phenomenom usually coupled with cell motility (Mattila and Lappalainen, 2008).
(23) Metabolic activity decreased in cells that were given Vc2CBMTD in serum-free medium (absence of FCS), compared to cells that were given PBS only. Cell morphology of Vc2CBMTD-treated cells was also observed to have changed, with cells appearing more rounded (a sign of cell arrest) when compared to the control. Interestingly, cell metabolic activity and phenotype appeared to be reversed with the addition of 2 mM sialyllactose. Similar concentrations of sialic acid can also be found in FCS, and this may be the reason why Vc2CBMTD-treated cells remained viable after 3 days compared to Sp2-treated cells, which appear to be unaffected by the presence of FCS, at least at the high dose of CBM used in the assay (FIG. 2). Alternatively, it may be that free sialyllactose in the medium compete for the glycan binding sites of Vc2CBMTD, thus allowing A549 cells to recover and proliferate.
(24) As for Sp2CBMTD-treated cells, there was also a slight (but not significant) decrease in metabolic activity. However, the lack of significance is likely due to the low concentration administered to the cells.
(25) There was a slight recovery of cell growth in the presence of sialyllactose compared to Vc2-treated cells, but due to the nature of Sp2CBMTD, which is also known to stimulate pro-inflammatory mediators, it is likely that the phenomenon observed in FIG. 2, could be due to its immunomodulatory role. It has been demonstrated that Sp2CBMTD increased a number of inflammatory mediators such as IFN-γ, VEGF, IP-10 and IL-8 and other cytokines that are known to be important in neutrophil and macrophage targeting (Govorkova et al (2015). This feature may be ideal for targeting the immune system to cancer cells. As for TcTs-treated cells, the CBM-like domain had little effect on cell metabolism and morphology.
EXAMPLE 2
(26) Wound Healing and Proliferation Assay of Cancer Cell Lines
(27) Method:
(28) All CBM40 proteins were prepared as described in Connaris et al (2014).sup.1. The following cancer cell lines, A549 (human lung), HeLa (cervical), MDA.MB.231 and MCF-7 (breast), and SW620 and SW480 (colon) were purchased from ATCC. All cells with the exception of HeLa cells were maintained in 10% FBS, DMEM (high glucose with 1% Penicillin/Streptomycin) and incubated at 37° C. and 5% C02. HeLa cells expressing GFP restricted to the nucleus (using IncuCyte NucLight Green Lentivirus Reagent Cat No. 4626 Essen Biosciences) were maintained in complete medium containing 0.5 μg/ml Puromycin. For wound healing assays, 24 well plates (Nunc) were seeded with cells (2.5×10.sup.5 cells/well) and maintained in 500 μL of their respective maintenance medium and incubated at 37° C. and 5% CO.sub.2 for 24 hrs. Wells were washed with 500 μL of serum-free DMEM, prior to the wound/scratch creation. A wound/scratch was created as a straight line down the middle of the well (running from the top to bottom of the well) in each well using a sterile 200 μL pipette tip (Axygen). Wells were then washed with 500 μL of serum-free DMEM prior to the addition of 250 μL of appropriately diluted CBMs, also prepared in serum-free DMEM. Serum-free medium was also included to wells as a control. Plates were incubated at 37° C. and 5% CO.sub.2 for 10-30 mins before a further addition of 250 μL of 4% FBS, DMEM to each well (2% FBS final concentration) so that the final CBM concentration ranged from 20-200 μg/ml. The IncuCyte ZOOM (Essen BioScience) apparatus was used to collect time-lapse images of each well. This was set to collect three images of the wound/scratch in each well every 1 hr for 72 h. For data collection, the IncuCyte ZOOM 2016 software was used to measure and collate wound width measurements for each image (3× wound measurements were performed manually). For the cell proliferation assay, GFP-labelled HeLa cells were grown in 24 well plates over 72 h in the presence or absence of mCBM40s. Cell proliferation is monitored by analysing the occupied area (percentage of confluence) of cell images over time using the IncuCyte ZOOM apparatus. Cell proliferation is directly proportional to increase in confluence.
(29) Results:
(30) FIGS. 5 and 6 illustrate the results of a wound-healing assay measuring the effect of different hexameric mCBM40s against different cancer cell types A549 and HeLa. The results indicate that hexameric CBM40s Vc2CBMTD and Sp2CBMTD significantly inhibited wound closure in both cancer types after 72 h compared to cells that were left untreated. Multimeric CBMs also inhibited wound closure in other cancer types that displayed differential expression of sialylated receptors. The cell growth of both breast and colon cancers were found to be inhibited in a dose-dependent manner (FIG. 7 illustrates the results from the highest dose of mCBM40s used against two metastatic cell types of breast and colon cancer). A difference in potency was observed between the highly metastatic and lowly metastatic breast cancer cell lines MDA.MB.231 and MCF-7. Multimeric CBMs were more potent against the MDA.MB.231 cell line. The potency did not differ much between the colon cancer cell lines SW620 and SW480 despite different glycosylation profiles. The percentage of wound covered for the two colon cancer cell lines and the low-grade breast cancer cell line after 72 hrs was low (between 30% to 40%).
(31) The inhibition of wound healing was due to the lack of cell proliferation over time as shown in FIG. 8, using GFP-labelled HeLa cells that demonstrated reduced cell growth over time. The anti-proliferative effect of mCBM40s was also observed with a tetrameric version mCBM40, Vc4CBM in both A549 and HeLa cells (FIG. 9).
(32) To determine whether the anti-proliferative effect is a result of the multimeric nature of engineered CBMs, A549 and HeLa cell lines were treated with the monomeric versions of Vc- and Sp-based CBM40s (VcCBM, SpCBM, 100 μg each) and left to incubate for up to 72 h. The data was then compared with that of their multimeric counterparts. FIG. 10 illustrates the results of a wound healing assay of A549 and HeLa cell lines after treatment with CBMs. At the dose used, multimeric CBMs had a significant effect on the rate of wound closure.
(33) Furthermore, the non-CBM40 component of mCBM40s, that is, the trimerisation domain (TD), was also tested in a scratch assay to determine if this domain may contribute to the anti-proliferative effect seen in the scratch assays. FIG. 11 demonstrates that the TD domain, when given at the same dose as the CBMs, does not appear to affect the cell growth or proliferation of A549 or HeLa cells, as the percentage and rate of wound closure was identical to untreated cells.
(34) To establish if anti-proliferative activity of CBM proteins can be reversed after a wound scratch assay, a wash-off experiment was attempted. After 72 h incubation, treated wells were washed twice with 0.5 mL of 2% FBS, DMEM (high glucose, 1% Penicillin/Streptomycin). The same medium (0.5 mL) was then added to each well and incubated for a further 72 h. The EVOS FL (ThermoFisher Scientific) apparatus was used to manually collect three images of the wound/scratch in each well at 0 hrs and 72 hrs. Image J software was used to manually measure and collate wound width measurements for each image (Image J measurement tool was calibrated against a scale bar for the EVOS FL). After removal of mCBM40s, cells were clearly seen to migrate and/or proliferate into the wound (FIG. 12). The rate and percentage of wound closure was observed to revert to “normal”/control back after the wash off. The rate of wound closure for VcCBM2TD after wash off was observed to be slightly faster than that of the control condition after 72 hrs. These observations suggest that mCBM40s can modulate the cell response to cancer by interacting with sialylated cell surface receptors.
(35) Migration Assay.
(36) Method:
(37) A Boyden chamber assay was selected to determine if cellular migration is interrupted by multimeric CBM40s. The Boyden chamber assay (8 μm, fluorometric format) was prepared as described in manufacturer's instructions (Cell Biolabs CytoSelect 96-well cell migration assay). Final CBM concentration ranged from 20-200 μg/ml.
(38) Results:
(39) FIG. 13 illustrate the results from the migration assay, which indicate that the CBMs tested inhibited migration of A549 cells. To eliminate the possibility that the lack of migration may be attributed to agglutination of A549 cells by mCBM40s when added directly to cell suspension, an agglutination assay was also performed using a spectrophotometric assay (Hwang et al, 1974).sup.2. The rate of absorbance/sedimentation of A549 cells over time did not differ greatly between the conditions (FIG. 14, A). This was further validated with a longer duration agglutination assay and a rate of absorbance/kinetics assay (FIGS. 14, B, C and D). This suggests that no agglutination of A549 cells (at 0.8×10.sup.6 cells/mL) in the presence of 1 mg/mL mCBM40 occurs. The inhibition of A549 cell chemotaxis can, therefore, be attributed to the direct inhibition of cellular migration and not cellular agglutination.
(40) Cell Apoptosis, Viability and Cytotoxicity Assay.
(41) Method:
(42) To determine if multimeric CBMs cause cell apoptosis, the Promega ApoTox-Glo™ Triplex Assay was used. This assay was performed using A549 cells. 10,000 cells per well were seeded 24 hrs prior to experiment. CBMs and controls were added to wells to a final volume of 100 μL with final CBM concentrations of 0.1-1 mg/ml. Cells were cultured in conditions for test exposures of 4 hrs, 24 hrs, 48 hrs and 72 hrs (FIG. 8).
(43) Results:
(44) After 48 hrs, Vc2CBMTD (at 400 μg) showed elevated luminescence signal corresponding to activation of caspase 3 (a hallmark of apoptosis). All the other conditions at 4 hrs, 24 hrs and 48 hrs showed similar luminescence levels to that of the untreated cells (FIG. 11). The fluorescence signal for the cleaved AFC is lower in CBM conditions after 48 hrs (FIG. 12). This might be due to either reduced permeability of the substrate, cells entering into a dormant state and/or cell cycle arrest. During most cytotoxic events, viability and cytotoxic measures will be inversely proportional; a reduction in viability without an increase in cytotoxicity is seen for compounds/drugs that alter normal cell division (i.e. cell cycle arrest) without producing membrane integrity changes. A number of the CBMs showed a decreased level of fluorescence for R110 indicating low levels of cytotoxicity (FIG. 13). Results from this cell viability, cytotoxicity and apoptosis experiment suggest that in the presence of mCBM40s, A549 cells are in cell cycle arrest or a state of dormancy.
(45) Discussion
(46) The results identify that CBMs (Vc4CBM, Vc2CBMTD and Sp2CBMTD), but not the non-CBM component (TD), inhibit cellular behaviours required for wound healing (proliferation and migration) and are effective against multiple cancer types as indicated by experiments using breast, colon, cervical and lung cells. Agglutination of the A549 cell line was not observed. The inhibition of both migrating and proliferating cells in the wound-healing assay can be reversed via a wash-off. The results obtained from the Promega ApoTox-Glo™ Triplex Assay suggest that CBMs cause cell cycle arrest/or dormancy in the A549 cell line.
References for Example 1
(47) Astarita, J L. et al., (2012). Frontiers in Immunology 3:283. Connaris, H et al., (2009). Journal of Biological Chemistry 284: 7339-7351. Connaris, H. et al., (2014). PNAS 111:6401-6406. Govorkova, E A et al., (2015). Antimicrobial Agents and Chemotherapy 59:1495-1504. Kato, Y. et al., (2005) Tumour Biology 26: 195-200. Mattila, P K. and Lappalainen, P. (2008). Nature Reviews Molecular Cell Biology 9: 446-454. Müthing, J. et al., (2002). Glycobiology 12: 485-497. Ochoa-Alvarez, J A. et al., (2012). PLoSONE 7:e41845. Schacht, V. et al., (2005). American Journal of Pathology 166: 913-921. Shibahara, J. et al., (2006). Virchow Arch. 448:493-499. Yau, T. et al., (2015) Molecules 20: 3791-3810. Zwierzina, H. et al., (2011) European Journal of Cancer 47: 1450-1457.
References for Example 2
(48) [1]. Connaris H, Govorkova E A, Ligertwood Y, Dutia B M, Yang L, Tauber S, Taylor M A, Alias N, Hagan R, Nash A A, Webster R G, Taylor G L. 2014. Prevention of influenza by targeting host receptors using engineered proteins. Proc Natl Acad Sci USA 111:6401-6406. [2]. Hwang, K. M., Murphree, S. A. and Sartorelli, A. C., 1974. A quantitative spectrophotometric method to measure plant lectin-induced cell agglutination. Cancer Research, 34(12), pp. 3396-3402.
